Over 15 million women suffer from urinary tract infections (UTI) annually in the U.S., ~85% of which are caused by uropathogenic Escherichia coli (UPEC). 20-40% of patients suffer from multiple and/or chronic recurrences and increasingly are left with few treatment options other than costly long-term antibiotic prophylaxis Indiscriminate use of antibiotics is leading to increased resistance to first-line empiric therapie such as trimethoprim-sulfamethoxazole and the undesirable use of fluoroquinolones for empiric treatment of UTI. Catheter-associated UTI (CAUTI) further exacerbates this problem. Consequently, multi-drug resistance is rising rapidly. Thus, there is a critical need for new therapeutics to better treat and prevent chronic recurrences. There are two bladder associated niches for UPEC during acute UTI: the bladder tissue and the luminal space/urine. FimH, the type 1 pilus adhesin mediates UPEC colonization and invasion of human and murine bladder epithelial cells (BECs). Murine models indicate that after invasion, UPEC can subvert innate expulsion mechanisms and escape into the cytoplasm where rapid bacterial replication results in the formation of intracellular bacterial communities (IBC) comprised of 104-105 bacteria. IBCs are transient and upon IBC maturation bacteria disperse from the biomass, filament, and spread to neighboring BECs, re-initiating the IBC cycle. This cycle potentiates the establishment of infection allowing the expansion of the bacterial population in a sequestered habitat protected from host immune and antibiotic clearance. Bacterial colonization of BECs, IBC formation, filamentation and biofilm formation on urinary catheters (also seen in mouse models) are all processes seen in human disease. Further, the fimH gene is under positive selection in UPEC clinical isolates consistent with its role in UTI. Murine models and human clinical studies show roles for the same specific cytokines and TLR4 signaling in UTI. In humans, UTI ranges from asymptomatic bacteriuria to acute self- limiting infection to chronic/recurrent UTI. Murine models mimic these disease outcomes and reveal that the nature of host response dictates whether a UTI resolves or develops into long-lasting chronic/recurrent UTI. This grant proposal will use a panel of virulent clinical isolates including a multi-drug resistant strain that has spread globall, to focus on a direct experimental investigation of common but complex clinical problems associated with more severe UTI and/or recurrence. Aim 1 investigates mechanisms by which superinfection of UPEC leads to the development of chronic/recurrent infection. Aim 2 investigates catheterization, which may alter the pathogenesis and may trigger the recurrence of infection in patients with a history of UTI. Aim 3 investigates the efficacy of potent small molecular weight compounds called mannosides, which block FimH function, to treat and prevent UTI in the context of these complicating factors. The proposed experiments will elucidate how sequential infection and catheterization affect the course of UTI and aid in better clinical management and the development of new therapeutics for combating this prevalent infection.